1. Field of the Invention
This invention is related in general to patch-clamp electrodes for electrophysiological testing of cells. In particular, the invention concerns a method for the manufacture of poly-dimethylsiloxane (PDMS) electrodes capable of forming high electrical resistance patch clamp recordings suitable for measurements in automated, repeatable, parallel experiments.
2. Description of the Related Art
Conventional voltage clamping techniques used to conduct electrophysiological tests on a membrane assess electrical activity on the membrane by measuring current or voltage changes produced in response to exposure to various test stimuli. Typically, the membrane is pierced with two microelectrodes connected to an amplifier capable of recording current or voltage variations in response to stimuli such as voltage step changes, the application of compounds, or mechanical stimulation.
Similarly, using patch clamping techniques, the membrane potential can be held constant while the current flowing through the membrane is measured to detect ion-channel activity that corresponds to changes in the membrane's conductance. Instead of using sharp microelectrodes to puncture the membrane and penetrate the cell, like in traditional voltage clamping, patch clamping uses a micropipette with a heat-polished tip of about 1 to 5 micron in diameter that is physically sealed to a “patch” on the membrane. The same pipette is used continuously for both current passing and voltage recording. For the most part, patch clamping is used either with a whole-cell or a single-channel mode of operation. In whole-cell patch clamping, the membrane at the tip of the pipette is ruptured to produce electrical continuity between the electrolyte in the pipette and the interior of the cell. Thus, total membrane current or voltage is measured. In single-channel patch clamping, the integrity of the membrane at the tip of the pipette is preserved. Accordingly, the recorded current is only the current flowing through the patch of the membrane enclosed by the tip of the pipette. Since this area is very small, there is a good chance that only one or a small number of ion channels may be in the membrane patch, and individual ion-channel currents may be recorded.
In both types of patch-clamp techniques, when the tip of the pipette is pressed against the cell membrane, the interior of the pipette is isolated from the extracellular solution by the seal that is formed between the tip of the pipette and the membrane. If the electrical resistance of the seal is sufficiently large, negligible current can leak across the seal and good measurements are obtained. Thus, any leakage of current through the seal is undesirable and the creation of a high-resistance seal (in the order of giga-ohms) is crucial for good results.
The basic design of a patch-clamp circuit 10 is illustrated in FIG. 1. A sensitive current-to-voltage converter 12 is fabricated using a high-megaohm (or giga-ohm, used for single-channel recordings) resistor 14 and an operational amplifier 16. A patch-clamp micropipette (only the tip 18 is shown) is connected to the negative input 20 and the control voltage 22 (Vcom) to the positive input 24 of the operational amplifier 16. The tip 18 of the pipette is adhered to the membrane of a test cell 26 immersed in a grounded electrophysiological bath 28 in a test chamber 30, such as to form a seal between the cell membrane and the glass pipette tip. Since the operational amplifier 16 has extremely high gain, the potential at the negative input 20 is forced to follow the potential Vcom established at the positive input 24. All current flowing in the micropipette also flows through the resistor 14. This current is proportional to the voltage across the resistor 14, and it is measured at the output 32 of the differential amplifier 34.
In order to improve the seal resistance for patch-clamp recordings, investigators have focussed on processes for improving the adhesion of conventional glass and quartz pipettes to the biological membrane. For example, Kiminori (Japanese Publication No. 4338240) taught that plasma treatment of the glass tip of a conventional micropipette improves the patch-clamp seal. New materials and patch-clamp seal geometries have also been investigated. For instance, Cytion's International Application No. PCT/IB98/01150 describes a perforated partition with multiple holes to form a plurality of patch-clamp seals between intra and extracellular compartments. In particular, K. G. Klemic et al. of Yale University have discovered that an electrode formed with silicone polymers (especially PDMS), in the form of a planar partition with multiple openings, is particularly suitable for producing giga-ohm patch-clamp seals with cell membranes.
The efficient construction of such a partition with apertures capable of providing effective patch clamps in repeatable and durable implementation remains a challenge in the art. This invention is directed at a simple and very effective process for manufacturing such a polymeric patch-clamp electrode.